Chimeras are composite organisms comprising cells derived from more than one zygote. Experimental chimeras have been used to study cell-to-cell interactions and perform cell fate and lineage analyses during development (McLaren, Mammalian Chimeras. Cambridge University Press, Cambridge, England, United Kingdom, 1976). The use of cells isolated from very early embryos to produce chimeras can result in organisms that develop with a full complement of somatic tissues partially made up of descendents of the isolated cells. If the starting material includes early germ cells or their precursors, the resulting chimeras can produce gametes of both the donor and recipient genotypes. In addition, chimeras can be intraspecific (i.e. containing cells derived from two or more zygotes of the same species) or interspecific (i.e. containing cells derived from zygotes from at least two different species).
The efficiency of generating germline chimeras by repopulating the gonads with the desired donor PGCs can be enhanced by reducing the number of PGCs in the recipient organism. A number of approaches to reduce PGCs have been utilized with varying degrees of success. Continuous exposure to gamma irradiation (0.3-3.4 R/hr of 60Co for 20 days) resulted in the complete destruction of oocytes at a dosage level of 3.4 and 1.8 R/hr (Mraz & Woody, Radiation Res 54:63-68, 1973). However, hatching frequency was reduced at levels of 0.9 R/hr or higher. The application of continuous low-level gamma irradiation to reduce endogenous PGC numbers is limited due to the relatively small numbers of eggs that can be exposed at any one time, the long period of exposure required, and also the potentially teratogenic effects of the irradiation itself.
Short-term exposure to a gamma source has also been attempted (Carsience et al., Development 117:669-75, 1993; Thoraval et al., Poultry Sci 73:1897-1905, 1994; Maeda et al., Poultry Sci 77:905-07, 1998). In these studies, unincubated eggs were exposed to 500-700 rads just prior to the injection of stage X blastodermal or area pellucida cells. The incidence of germline chimerism following short-term gamma irradiation was highly variable. The basis for the inconsistent results was ascribed to “donor cells being injected into an inappropriate location . . . ” (Carsience et al., Development 117:669-75, 1993).
Attempts to sterilize recipient embryos using ultraviolet (UV) light have also been described (Reynaud, J Embryol Exp Morphol 21:485-507, 1969; Reynaud, J Roux's Arch Devel Biol 179:85-110, 1976; Aige-Gil & Simkiss, Br Poul Sci 32:427-438, 1991). Aige-Gil & Simkiss concluded “it is not possible to irradiate the germinal crescent, particularly at stage 4 of incubation, without inducing major abnormalities”. The degree of sterility appeared to be positively correlated with developmental abnormalities, thus limiting the practical use of UV-light as a means to reduce endogenous PGC.
The generation of germ line chimeras produces several potential benefits both to mankind and to the various avian species themselves. Germ line chimeras can be used as a source of gametes with desirable characteristics, which can then be used in conjunction with breeding programs to augment the avian gene pool. The ability to more easily produce gametes of particular avian species would be useful to the avian veterinary and poultry production fields. For endangered species such as the whooping crane, it would be extremely useful to have a ready supply of male spermatozoa. For commercial birds such as turkeys, it would be desirable to more quickly and economically produce male spermatozoa. For meat-producing flocks, it is desirable to have ways to increase the ratio of male birds in the flock. As such, there is a need for new ways to obtain avian spermatozoa.
Accordingly, there remains a long-felt and continuing need for ways to increase the efficiency of the production of germ line chimeras in avians. The presently disclosed subject matter addresses this and other needs in the art.